Voltage-gated sodium channels (VGSCs) generate and propagate electrical activity in excitable cells. The precise dynamics of VGSC transitions among closed, open, and inactivated states are essential for brain, nerve, heart, and muscle function, as documented by a wide range of clinical sodium channelopathy disorders. While the VGSC alpha subunit in isolation comprises the channel's pore, its voltage sensors, and its inactivation machinery, interaction with fibroblast growth factor homologous factors (FHFs) has large and complex effects on the dynamics of VGSC inactivation. Indeed, FHF mutations are a cause of human spinocerebellar ataxia and cause defects in neuronal intrinsic excitability. While all FHF isoforms bind VGSCs, they differ in their abilities to modulate VGSC fast inactivation and to induce a newly characterized long-term inactivated channel state. This application proposes experiments to expand analysis of FHF neuronal functions and physical mechanisms of FHF- induced VGSC modulation. AIM I: Our overall biological hypothesis is that differential expression of FHF isoforms in different neurons and their subcellular compartments acts to determine excitation and conduction properties of cells. We will test: (I-A) Does the relative abundance of different long-form FHFs (A-type FHFs, FHF4B) at the axon initial segment specify the excitation properties of a neuron? (I-B) Does somatodendritic membrane localization of A-type FHFs act to limit sodium action potential backpropagation during repetitive firing of neurons? (I-C) Are long-form FHFs not required for, and potentially deleterious to, action potential axonal conduction? AIM II: A clearer understanding of physical mechanisms for VGSC modulation by FHFs will provide further insight into VGSC conformation dynamics and suggest rational approaches for designing therapeutics for managing disorders of hyperexcitability, such as epilepsies and arrhythmias. We will test: (II-A) Does the long- term inactivation particle at the distal N-terminus of A-type FHFs dock within the cytoplasmic cavern of a VGSC? (II-B) Do the cationic residues in the docked A-type FHF inactivation particle act as a shield against sodium ion conduction? (II-C) How does a cationic region near the ?-trefoil core of FHFs modulate VGSC fast inactivation?